© 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 866-875 doi:10.1242/dev.140368

RESEARCH ARTICLE The E3 ligase Nedd4/Nedd4L is directly regulated by microRNA 1 Jun-yi Zhu1, Amy Heidersbach2,3, Irfan S. Kathiriya2,4, Bayardo I. Garay2,4, Kathryn N. Ivey2,3, Deepak Srivastava2,3, Zhe Han1,* and Isabelle N. King2,3,*

ABSTRACT homologous to the E6-AP C-terminal (HECT) domain (reviewed by miR-1 is a small noncoding RNA molecule that modulates Rotin and Kumar, 2009). Although its regulation is relatively unexplored, expression in heart and skeletal muscle. Loss of Drosophila miR-1 Nedd4 activity is governed by multiple post-translational modifications produces defects in somatic muscle and embryonic heart and an array of co-factors (reviewed by Yang and Kumar, 2010). development, which have been partly attributed to miR-1 directly MicroRNA 1 (miR-1) is evolutionarily conserved from flies to targeting Delta to decrease Notch signaling. Here, we show that humans and is crucial for normal cardiac development and function overexpression of miR-1 in the fly wing can paradoxically increase (Heidersbach et al., 2013; Ivey et al., 2008; King et al., 2011; Kwon Notch activity independently of its effects on Delta. Analyses of et al., 2005; Sokol and Ambros, 2005; Wei et al., 2014). Drosophila potential miR-1 targets revealed that miR-1 directly regulates the miR-1 negatively regulates the Notch ligand Delta in the developing 3′UTR of the E3 Nedd4. Analysis of embryonic and Drosophila wing, producing a loss of Notch activity (Kwon et al., adult fly heart revealed that the Nedd4 protein regulates heart 2005). Using a GAL4-UAS-based system in the fly wing, we identified development in Drosophila. Larval fly hearts overexpressing miR-1 genetic pathways that intersect with miR-1 (King et al., 2011). This have profound defects in actin filament organization that are partially system is particularly adept at detecting interactions between miR-1 rescued by concurrent overexpression of Nedd4. These results and the Notch pathway due to the importance of Notch signaling in indicate that miR-1 and Nedd4 act together in the formation and actin- wing-vein patterning. Specifically, increased Notch signaling results dependent patterning of the fly heart. Importantly, we have found that in a loss of vein structures, whereas decreased Notch signaling results the biochemical and genetic relationship between miR-1 and the in thickened, tortuous veins (de Celis and García-Bellido, 1994). mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved Unexpectedly, we observed a progressive loss of vein structures as in the mammalian heart, potentially indicating a role for Nedd4L in miR-1 levels rose, suggesting that, in certain contexts, miR-1 can mammalian postnatal maturation. Thus, miR-1-mediated regulation promote Notch signaling. Given this unexpected result, we speculated of Nedd4/Nedd4L expression may serve to broadly modulate the that in addition to its effects on Delta, miR-1 also governs the activity trafficking or degradation of Nedd4/Nedd4L substrates in the heart. of a negative regulator (or regulators) of the Notch pathway. To identity this miR-1 target, we focused on a particular vein phenotype KEY WORDS: miR-1, Nedd4, Nedd4L, Nedd4-2, Heart that results in a truncated third long vein (L3).

INTRODUCTION RESULTS Notch signaling regulates many aspects of cell differentiation and Overexpression of miR-1 in the wing can increase Notch specification. In the developing Drosophila wing, Notch cooperates signaling with the Hedgehog (Hh) pathway to form and pattern the anterior- Using the decapentaplegic-GAL4 driver (dpp-GAL4) system, we posterior (AP) boundary (Casso et al., 2011). Several E3 ubiquitin expressed Drosophila miR-1 specifically in the AP organizer of the ligases assure that Notch and its ligands properly migrate from the cell wing imaginal disc (dpp>miR-1) (Brand and Perrimon, 1993; King surface to various endocytic compartments for activation (e.g. Deltex, et al., 2011). Capitalizing on the temperature responsiveness of the Mindbomb1). By contrast, the E3 ubiquitin ligase Nedd4 (neural GAL4-UAS system, we placed the dpp>miR-1 line at 20°C or 22°C precursor cell-expressed, developmentally downregulated 4) negatively and scored the wings for changes in vein patterning and L3/L4- regulates Notch by directing it to the lysosome (Ingham et al., 2004; intervein distance. As miR-1 expression increased, the wing veins reviewed by Rotin et al., 2000; Sakata et al., 2004). Nedd4, along with thickened and intervein distance decreased progressively, consistent its family of proteins, has a phospholipid-binding , two to with miR-1 repressing the Notch ligand Delta in the AP organizer. four WW domains that recognize substrates, and a catalytic domain Unexpectedly, at 22°C, when miR-1 expression is highest, a significant number of flies were missing the distal region of L3, a wing phenotype similar to that produced by increased Notch 1Children’s National Medical Center, Washington, DC 20010, USA. 2Gladstone signaling (Fig. 1A, lower panel; Fig. 1B). Institute of Cardiovascular Disease, San Francisco, CA 94158, USA. 3Department of Pediatrics, University of California, San Francisco, CA 94143, USA. 4Department Next, we focused on the molecular mechanism of this truncation. of Anesthesia and Perioperative Care, University of California, San Francisco, CA To determine whether the distal loss of L3 under conditions of high 94143, USA. miR-1 expression reflected increased Notch signaling, we genetically *Authors for correspondence ([email protected]; crossed alleles of positive and negative regulators of the Notch [email protected]) pathway. All parental lines had wild-type L3-vein morphology (data not shown). When mated to dpp>miR-1 flies, fly lines containing J.Z., 0000-0002-4517-101X; D.S., 0000-0002-3480-5953; Z.H., 0000-0002- 5177-7798; I.N.K., 0000-0002-3182-7540 loss-of-function alleles of positive regulators of Notch (i.e. Notch, deltex, Suppresssor of Hairless) showed a variable loss of intervein

Received 1 June 2016; Accepted 10 January 2017 distance, indicating a genetic interaction with miR-1 (Fig. S1A). DEVELOPMENT

866 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 1. Misexpression of miR-1 in the anterior-posterior organizer causes a derepression of the Notch pathway. (A) Representative wings (left) and schematic of wing phenotypes are shown (right). Shaded areas indicate regions of dpp- GAL4 activity, such that miR-1 is overexpressed in the dpp>miR-1 fly line. (B) Quantification of dpp>miR-1 progeny lacking the distal region of L3 at different temperatures. The UAS-GAL4 system allows titration of expression by temperature manipulation. n, number of progeny scored. (C) A simplified schematic of Notch repression. In the nucleus, Notch associates with its DNA-binding partner Suppressor of Hairless [Su(H)] to activate downstream target , some of which repress Notch signaling in a negative-feedback loop. (D) Percentage of progeny with a truncated L3 by genotype when crossed with the dpp>miR-1 line at 18°C. At this temperature, L3 forms normally in the control dpp>miR-1 line and loss of the L3-L4 intervein distance predominates. n, number of progeny scored. *P<0.001. Gro, Groucho; Gsc, Goosecoid; H, hairless.

However, none of these alleles generated flies that lacked the distal formation. From these results, we concluded that the distal loss of L3 region of L3, suggesting that reductions in the gene dose of Notch results from de-repression of the Notch pathway and reflects an effectors were not responsible for the shortening of L3. epistatic relationship between miR-1 and Notch repressors.

Dysregulation of Delta ubiquitylation or expression does not Truncation of L3 in dpp>miR-1 flies is not due to reduce L3 length phosphorylation of Groucho by receptor tyrosine kinases To determine whether dysregulation of Delta caused the loss of L3 Loss-of-function alleles of groucho, hairless or goosecoid could structures, we manipulated the relative amounts of active Delta with impair L3 formation by genetically interacting with a separate mutants of mindbomb1 (mib1) and UAS lines expressing wild-type genetic pathway that is also required for vein formation in the wing. or dominant-negative Delta (UAS-Delta and UAS-DeltaD/N, For example, epidermal growth factor receptor (EGFR) signaling respectively). Mib1 facilitates the activation of Delta through modulates Notch activity through phosphorylation of Groucho ubiquitylation and subsequent endocytosis of the ligand (Itoh et al., mediated by tyrosine receptor kinase (Hasson et al., 2005). To 2003). Interestingly, reduced levels of mib1 did not affect the determine whether the length of L3 is sensitive to EGFR activity, we dpp>miR-1 phenotype, suggesting that the loss of L3 is not sensitive tested effectors of EGFR signaling, both positive [e.g. Spitz (spi), to the level of Mib1 expression in this system (Fig. S1B). Likewise, Star (S), Rhomboid (Rho), Vein (vn) and Roughoid (ru)] and when crossed with the dpp>miR-1 line, neither the UAS-Delta nor the negative [e.g. Knot (also known as Collier) (kn), Argos (aos)]. UAS-DeltaD/N lines produced offspring with L3 truncation (Fig. S1B). Isolated alleles of S, Rho, ru, spi, vn, aos,orkn were crossed with the Wings overexpressing both miR-1 and DeltaD/N hadathickenedand dpp>miR-1 line. Scoring of the genetically relevant progeny did not tortuous L3; however, the most distal region of L3 could not be reliably reveal a genetic interaction between the EGFR pathway and miR-1 visualized, as the wings were globally deformed (data not shown). (Fig. S2). However, their interaction was genetically enhanced These results suggest that miR-1-mediated reduction of Delta or gross when fly lines were deficient in ED207 and BSC289. These lines are perturbation of Delta expression is insufficient to inhibit L3 formation. haploinsufficient at both the Rho and ru loci, and produce a variation in the intervein distance between L3 and L4 (the fourth Reductions in the dose of Notch co-repressors lead to L3 long vein). However, in no instance did a reduction in gene dose of truncation positive or negative regulators of EGFR signaling cause loss of the The Notch receptor associates with its DNA-binding partner distal region of L3, nor did alleles of constitutively active EGFR Suppressor of Hairless [Su(H)] to activate several downstream (ElpB1/ellipse) affect L3 formation (Baker and Rubin, 1989) genes, some of which form a negative-feedback loop (Fig. 1C). When (Fig. S2). Thus, L3 shortening does not depend on EGFR we crossed transgenic lines containing loss-of-function alleles of signaling or EGFR-mediated phosphorylation of Groucho. Notch co-repressors (i.e. groucho, hairless or goosecoid)withthe dpp>miR-1 line, we observed an enhanced loss of intervein distance miR-1 regulates the Notch pathway through conserved and a truncated L3 – phenocopying the wings of dpp>miR-1 flies targeting of Nedd4 raised at 22°C (Fig. 1D). These results were obtained at 18°C, where To explore the possibility that miR-1 directly downregulates miR-1 expression is below the usual threshold for suppression of L3 groucho, hairless or goosecoid, we interrogated the Targetscan DEVELOPMENT

867 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368 fly database (www.targetscan.org) and determined that no probable be generalized beyond the interaction of miR-1 and Nedd4 and that miR-1 sites exist in the 3′UTRs of these genes. By contrast, Su(H), Nedd4 family members have non-redundant functions in our mib1 and Nedd4 each contain one evolutionarily conserved miR-1 dpp>miR-1-based system. site (data not shown). Interestingly, in Drosophila Schneider 2 (S2) cells transiently transfected with miR-1, followed by Reduced Nedd4 activity is due to a functional miR-1-binding immunoprecipitation of Argonaut1 (Ago1) and quantitative site in the 3′UTR of Nedd4 RT-PCR, Nedd4 mRNA was enriched (Easow et al., 2007). These The 3′UTRs of fly Nedd4 and murine Nedd4l are bioinformatically results support the hypothesis that miR-1 and Nedd4 mRNA predicted to contain miR-1-binding sites (Fig. 2B). To assess the physically associate with each other within a miRNA-effector biological relevance of these putative sites, we cloned the 3′UTRs of complex. These findings prompted us to focus on a possible genetic Nedd4 or Nedd4l into luciferase-reporter constructs. Introduction of or biochemical relationship between miR-1 and Nedd4. the miR-1 mimic into the murine C2C12 myoblast cell line Nedd4 ubiquitylates the PPSY motif in the intracellular domain significantly reduced luciferase activity, suggesting that miR-1 of Notch in a HECT domain-dependent fashion (Sakata et al., negatively regulates Nedd4 mRNA in mammalian cells (Fig. 2G, 2004). Ubiquitylation of this motif by Nedd4 or Suppressor of left). Deletion of the putative miR-1 seed sequence within the Deltex [Su(dx)] reduces Notch signaling by directing the receptor to predicted target site prevented the repressive effects, demonstrating late endosomes. Nedd4-like proteins are evolutionarily conserved that silencing is miR-1 dependent (Fig. 2G, right). Parallel studies from yeast to mammals. Nedd4 and its murine orthologue Nedd4L also performed in the C2C12 myoblast cell line using the Nedd4l 3′ (also known as Nedd4-2) share a common domain structure UTR produced similar results (see Fig. 6A), suggesting that the (Fig. 2A). Nedd4 localizes to the through its C2 regulation of Nedd4 and Nedd4l by miR-1 is evolutionarily domain and uses its WW domains to recognize substrate proteins. It conserved. negatively regulates Notch signaling by directing the Notch receptor towards endocytosis and lysosomal degradation. Interestingly, the Nedd4-null flies have abnormal heart specification and predicted miR-1 binding site in the 3′UTR of Nedd4 and Nedd4l is patterning that result in early lethality widely conserved (Fig. 2B). To determine whether the genetic and biochemical relationship We hypothesized that the loss of L3 structures is due to miR-1- between miR-1 and Nedd4 was relevant in tissues where miR-1 is mediated downregulation of Nedd4. To test this hypothesis, we endogenously expressed, we examined the cardiac phenotypes of analyzed alleles of Nedd4 in the wing assay at both 18°C and 20°C. the two Nedd4 mutant alleles (Nedd4T119FS and Nedd4MI07766)ina At 18°C, the dpp>miR-1 line had minimal miR-1 expression, and Hand-GFP (Han and Olson, 2005) background, which supports penetrance of the L3 truncation phenotype was low but measurable visualization of cardioblasts and pericardial cells in the developing with two Nedd4 alleles (Nedd4MI07766, 4.7%, n=103; Nedd4C153, fly heart. Analysis of these animals revealed that Nedd4 is necessary 2.3%, n=130; data not shown). At 20°C, where truncation of L3 is for normal heart formation in the embryonic period and first instar detected in the dpp>miR-1 line, all alleles of Nedd4 generated stage (Fig. 3). Homozygous null Nedd4 embryos were distinguished significant numbers of progeny with a shortened L3 (Fig. 2C, using a GFP-positive balancer and stained with Fig. S3). The weakest allele, Nedd4T119FS, also generated progeny antibodies against Mef2, a marker of somatic and heart muscle. We with L3 breaks (17%, n=54; data not shown). Thus, reducing the observed that most homozygous Nedd4 mutants displayed reduced Nedd4 dose enhances the effects of miR-1, resulting in loss of L3 numbers of cardioblasts and pericardial cells at stage 15, resulting in structures. To verify that the loss of Nedd4 activity was responsible gaps among the two rows of cardioblasts (Fig. 3A-C). Homozygous for the effects on L3 formation, flies harboring a wild-type Nedd4 mutants of either of the two alleles die around the late first-instar construct (UAS-Myc-Nedd4) (Myat et al., 2002) were crossed to the larval stage. Although cardiac structures such as ostia (hemolymph dpp>miR-1 line. For all alleles tested, concurrent overexpression of inflow tracts) still form, the number of cardioblasts is reduced, wild-type Nedd4 robustly reduced the number of progeny lacking reflected by fewer than four non-ostia cardioblasts in each the distal region of L3 (Fig. 2D). To determine whether an intact hemisegment (Fig. 3D-F). These results indicate that Nedd4 is HECT domain was necessary for rescue of the wing vein phenotype, necessary to generate the expected numbers of cardioblasts. we crossed a line containing a ubiquitin ligase inactive Nedd4 construct [UAS-Myc-Nedd4(C/A)] with the dpp>miR-1 line (Myat Misexpression of Nedd4 disrupts the patterning of et al., 2002). Again, we observed marked suppression of the wing cardioblasts and ostia cells in the adult fly hearts vein phenotype for all alleles except for Nedd4T119FS and Nedd4C153 As Nedd4 and miR-1 are both expressed in the developing fly heart, (Fig. 2E). These results suggest that the L3 truncation is attributable we speculated that miR-1 might modulate the absolute levels of to reduced levels of Nedd4, though an intact HECT domain is not Nedd4 within the fly heart. To determine the sensitivity of the fly essential for this effect. heart to Nedd4 activity, we overexpressed Nedd4 in the heart using twist-GAL4 or Hand-GAL4 drivers (Han and Olson, 2005). The genetic interaction between miR-1 and Nedd4 does not Interestingly, using these UAS-GAL4 systems (Brand and extend to other closely related E3 ubiquitin ligases Perrimon, 1993) to misexpress wild-type (UAS-Myc-Nedd4) and Like Nedd4, Su(dx) negatively regulates Notch. In wing discs, ubiquitin-ligase mutant forms of Nedd4 [UAS-Myc-Nedd4(C/A)], as Nedd4 can compensate for the loss of Su(dx), implying functional well as Nedd4-RNAi, within the developing mesoderm permitted redundancy (Wilkin et al., 2004). Therefore, we sought to determine the recovery of adult flies (Fig. 4). Close examination of first-instar whether miR-1 interacts genetically with other members of the larvae revealed mild defects in the arrangement of the four pairs of Nedd4 family [i.e. Su(dx), Smurf] using L3 morphology as a Tinman-positive cardioblasts separated by two pairs of ostial cells readout. Su(dx)32, but not Su(dx)KG02902 or Su(dx)2, generated flies (Fig. S4A,B). At 29°C, Hand>UAS-Myc-Nedd4, Hand>UAS-Myc- with a shortened L3. Similarly, SmurfKG07014, but not SmurfMI07104 Nedd4(C/A) and Hand>UAS-Nedd4-RNAi animals had mildly or Smurfs-160, genetically interacted with miR-1 to repress L3 reduced survival to eclosion and reduced adult life expectancy formation (Fig. 2F). These results indicate that L3 shortening cannot compared with control lines (n=60 for each genotype; Fig. S4C,D). DEVELOPMENT

868 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 2. Nedd4 genetically interacts with miR-1, resulting in a truncated L3. (A) A structural schematic of Nedd4 with the tested mutations Nedd4MI07766, Nedd4C153 and Nedd4EY00500 alleles are p-element insertions in upstream non-coding regions and are not depicted. Not drawn to scale. C2, Ca2+-dependent phospholipid binding domain; HECT, catalytically active ubiquitin ligase domain; WW, substrate selection and cellular localization domain. (B) Schematic of the 3′UTR of Nedd4 from various species; the conserved predicted miR-1 target sequence is highlighted in blue. (C) Number of flies lacking the distal region of L3 by genotype (performed at 20°C). *P<0.001. n, number of flies scored. (D) Genetic rescue studies. Graph shows the percentage of affected offspring for the noted crosses (performed at 20°C). The loss of L3 is less penetrant when wild-type Nedd4 is co-expressed with miR-1.*P<0.001. n, number of flies scored. (E) Genetic rescue studies as in D using a mutant form of Nedd4 lacking a functional HECT domain. Number of flies lacking the distal region of L3 by genotype (performed at 20°C). n, number of flies scored. In some genetic backgrounds, a functional HECT domain is not required for suppression of the vein truncation phenotype. (F) As in C. The Nedd4 family members Su(dx) and Smurf produce L3 truncation in selected genetic backgrounds. (G) Luciferase activity in transfected C2C12 (murine myoblast) cells of a reporter construct containing the 3′UTR of Nedd4 (left) or a reporter construct with a deletion of the predicted miR-1-binding element (right).*P≤0.05. (Bottom) Schematic showing base pairing of Nedd4 mRNA with miR-1. Blue font indicates the miR-1 seed sequence. Blue box indicates residues that were deleted to disrupt miR-1 sensitivity and tested (right-hand bars).

Altered Nedd4 expression caused pericardial cells to detach harboring the Hand>UAS-Myc-Nedd4, Hand>UAS-Myc-Nedd4 from mature fly hearts (C/A) or Hand>UAS-Nedd4-RNAi constructs. Individual hearts To determine whether these modest disruptions in the normal were fixed and stained with phalloidin to visualize the structure of patterning of first instar larvae resulted in morphological defects in the cardiac actin filaments. Pericardial cells were marked with the adult heart, we collected newly eclosed (adult) mutant fly hearts Hand-GFP; an anti-pericardin antibody labeled the extracellular DEVELOPMENT

869 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 3. Nedd4 is required for normal patterning of the Drosophila heart. (A-C) Stage 15 Nedd4-null embryos marked by a Hand-GFP (cytoplasmic) construct to visualize cardioblasts and pericardial cells (green), with the genotype indicated in the lower right-hand corner. Anterior is towards the left. (A′-C′) Embryos stained with antibodies against Mef2 (Drosophila myocyte enhancer factor 2), a muscle marker (red), compared with representative Nedd4T119FS and Nedd4MI07766 embryos with areas of reduced numbers of cardioblasts (yellow brackets). Genotypes as in A-C. (A″-C″) Overlay of A-C with A′-C′. (D-F) First-instar larvae with genotypes indicated in the bottom right-hand corner. Anterior is towards the left. (D′-F′) Higher magnification (20×) of the area highlighted by the yellow boxes in D-F, with ostial cells outlined by dashed lines and cardioblasts indicated by white arrows. Normally there are four cardioblasts between each set of four ostial cells; in Nedd4T119FS and Nedd4MI07766 mutant larvae, the localization and/or number of these cardioblasts is abnormal. n=6 per phenotype. matrix of the heart tube. Interestingly, compared with wild-type concurrent Nedd4 overexpression, we generated animals that controls, animals with supraphysiological expression of Nedd4 or concurrently overexpress miR-1 and Nedd4 or Nedd4(C/A) the catalytically inactive form of Nedd4 [Nedd4(C/A)] showed a [Hand>UAS-Myc-Nedd4+UAS-miR-1, Hand>UAS-Myc-Nedd4 dramatic increase in the number of detached pericardial cells, (C/A)+UAS-miR-1]. Animals were raised at 20°C and pertinent generalized disorganization of cardiac actin filaments (Fig. 4A-D) progeny evaluated. We did not recover adult animals when miR-1 and a loss of cardiac muscle-fiber density (Fig. 4E). This phenotype was overexpressed, regardless of Nedd4 expression, indicating that is strikingly similar to other cardiac extracellular-matrix mutants, overexpression of Nedd4 does not completely rescue abnormalities such as Lonely heart (loh) (Drechsler et al., 2013). To determine induced by excessive miR-1 expression. By contrast, we were able to whether pericardial cells detached in the context of abnormal detect viable larvae, suggesting that genetic rescue may occur at extracellular-matrix formation, we stained the flies for pericardin, a earlier developmental stages. To visualize larval hearts, individual type IV collagen that is crucial for maintaining cardiac integrity in animals were fixed and stained with phalloidin to visualize the Drosophila. Consistent with a role for Nedd4 for normal cardiac structure of the cardiac actin filaments. Overexpression of miR-1 extracellular-matrix formation, we observed increased pericardin universally resulted in profound disorganization of the actin deposition in flies overexpressing Hand>UAS-Myc-Nedd4(C/A), structures compared with wild type (Fig. 5B). Surprisingly, we which expresses catalytically inactive Nedd4 (Fig. 4C″). These found that, at L3, overexpression of Nedd4 and, to a lesser extent, results indicate that Nedd4 may help maintain the normal Nedd4(C/A) was able to reduce the myofibrillar disorganization myofibrillar structure of the heart tube and its associated induced by overexpression of miR-1. These results indicate that, in pericardial cells. We propose that some of its effects on heart larval heart, increasing Nedd4 expression can normalize actin fiber structure may be secondary to changes in pericardin deposition. organization induced by excessive miR-1 expression.

Changes in muscle fiber-associated actin filaments induced Nedd4L and miR-1 levels are inversely proportional in by overexpression of miR-1 can be rescued by increasing mammalian hearts expression of Nedd4 during larval stages These in vivo results prompted us to determine whether miR-1 and To determine whether abnormalities in heart structure or function Nedd4l interact genetically and biochemically in the mammalian induced by overexpression of miR-1 could be genetically rescued by heart. As with the luciferase assays testing the 3′UTR of Nedd4 for DEVELOPMENT

870 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 4. Misexpression of Nedd4 in cardioblasts results in actin-fiber disarray and abnormal pericardin deposition. (A-D) Newly eclosed adult flies harboring a Hand-GFP (cytoplasmic) construct to visualize pericardial cells (green) with muscle-associated actin filaments (red) and pericardin (blue). Genotype is indicated in the lower right corner. Anterior is towards the left. (A′-D′) Higher magnifications (20×) of areas outlined by the yellow boxes in A-D, demonstrating the normal arrangement of actin filaments (A′) versus the disarrayed actin fibers seen in Hand>UAS-Myc-Nedd4, Hand>UAS-Myc-Nedd4(C/A) and Hand>UAS- Nedd4-RNAi (B′-D′). (A″-D″) As for A′-D′ with the heart tube outlined (dashed line) where pericardin deposition was quantified. n=3 per phenotype. (E) Quantification of cardiac muscle-fiber density by genotype, normalized to wild type. ***P<0.01. (F) Quantification of pericardin staining in the heart tube area as outlined in A″-D″.*P<0.05. (G) Quantification of cardioblast number by genotype. miR-1 sensitivity, the 3′UTR of Nedd4l was also sensitive to miR-1 physiologically increase, Nedd4L levels would fall. To test this regulation (Fig. 6A). To determine whether the expression of Nedd4L hypothesis, we performed western blot analysis on whole-heart was inversely proportional to miR-1 levels in murine hearts, we took lysates obtained from wild-type mice at postnatal days 2 and 21 (P2 advantage of the fact that miR-1 levels rise after birth as part of the and P21) using anti-Nedd4L antibodies (Bethyl). Each postnatal transition from the fetal to the postnatal circulation (Fig. 6B). We period was represented by a minimum of five individual hearts, and postulated that during this postnatal period, as miR-1 levels the amounts of Nedd4L were normalized to the GAPDH loading DEVELOPMENT

871 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 5. Overexpression of Nedd4 is able to rescue miR-1-mediated actin fiber disarray in larval hearts. (A) Wild-type larval (L3) heart stained with muscle-associated actin filaments. (B) Overexpression of miR-1 in the fly heart using the Hand>UAS-miR-1 expression construct results in actin filament disorganization and collapse. (C) Concurrent expression of Nedd4 with miR-1 (Hand>UAS- Myc-Nedd4+UAS-miR-1) significantly normalizes actin fiber organization. (D) Concurrent expression of a ubiquitin ligase-deficient mutant of Nedd4 with miR-1 [Hand>UAS-Myc-Nedd4(C/A)+UAS-miR-1] improves actin fiber organization. (E,F) Overexpression of Nedd4 (Hand>UAS- Myc-Nedd4) or mutant Nedd4 [Hand>UAS- Myc-Nedd4(C/A)] in larval hearts has minimal effects on actin fiber organization. All larvae were raised at 29°C. n=6 for each genotype.

control. We found that the levels of Nedd4L expression were ∼2.5- positive effectors of the Notch receptor (e.g. Deltex) or fold higher in P2 hearts compared with P21 hearts. Based on these perturbations in Delta-mediated cis-inhibition, contributed to the results, we investigated whether Nedd4L expression was perturbed in de-repression of Notch in our wing-based assay system. miR-1 null (miR-1-1−/−;miR-1-2−/−) mice. We performed a targeted Our findings in the mammalian heart indicate that the genetic and analysis of data from RNA sequencing of heart lysates from late biochemical interaction between miR-1 and Nedd4l is physiologically embryonic (E18) miR-1 null (miR-1-1−/−; miR-1-2−/−) mice versus relevant and may provide developmental or tissue-specific regulation wild-type controls (Heidersbach et al., 2013). Consistent with a of Nedd4l in the myocardium. We speculate that the additional bands model in which miR-1 directly targets Nedd4l, miR-1-null hearts had observed on western blots of heart lysates using an anti-Nedd4L significantly increased levels of Nedd4l mRNA (Fig. 6D). To antibody might result from post-translational modifications, because determine whether this increase in Nedd4L RNA altered protein Nedd4L can autoregulate its stability through ubiquitylation of expression, we analyzed lysates of P2 murine heart cells by western its HECT domain (Bruce et al., 2008). Alternatively, they might blot (Fig. 6E). In wild-type P2 hearts, Nedd4L levels were low but represent heart-specific splice variants, because tissue-specific detectable. By contrast, miR-1-null mice had significantly increased isoforms of Nedd4L have been found in the heart and the liver Nedd4L levels at the predicted molecular weight, plus two additional (Chen et al., 2001; Fu et al., 2013). bands (n=3 per genotype), as seen in the wild-type hearts. Nedd4L Importantly, although miR-1-mediated reductions in Nedd4 levels in miR-1-null mice at P21 could not be determined, because the activity caused wing-vein phenotypes induced by Notch, miR-1- miR-1 null state is lethal by P7 owing to heart failure. In summary, mediated dysregulation of Nedd4L in the heart likely affects these results support the hypothesis that miR-1 regulates Nedd4l to proteins outside the Notch pathway. Indeed, protein microarrays affect the physiology of the mammalian heart. comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion DISCUSSION channels, whereas Nedd4 targets are enriched for signaling Unexpectedly, we found that overexpression of miR-1 in the pathways (Persaud et al., 2009). Thus, in the heart, where miR-1 anterior-posterior (AP) organizer of the wing disc results in a dose- and murine Nedd4L are both expressed, their genetic and dependent loss of L3 vein structures, consistent with de-repression biochemical interaction might influence the excitability and of Notch or weakening of a regulatory mechanism that dampens the connectivity of cardiomyocytes. Indeed, susceptibility to cardiac Notch signal. Using genetic techniques, we determined that the loss arrhythmias and sudden death in humans is associated with of the distal aspect of L3 could be phenocopied by reducing the gene six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, dose of Notch co-repressors or Nedd4; in the case of Nedd4, the KCNE1, KCNE2 and RYR2) (reviewed by Keating and Sanguinetti, regulation by miR-1 was direct. We propose an expanded model in 2001). Murine Nedd4L regulates the cell-surface densities of the which miR-1 expression in the AP organizer has complex effects on sodium channel, the voltage-gated type V alpha subunit (Scn5a) Notch signaling owing to its regulation of ligand availability and (Abriel et al., 2000; Rougier et al., 2005; van Bemmelen et al., receptor trafficking. As lower levels of miR-1 expression (18°C) 2004), the potassium voltage-gated channel, KQT-like subfamily caused wing-vein thickening and tortuosity, and higher levels member 1 (Kcnq1) (Jespersen et al., 2007; Krzystanek et al., 2012) (22°C) caused vein loss, Delta and Nedd4 may be differentially and the human Ether-a-go-go-related (KCNH2, previously hERG) sensitive to miR-1 regulation, although our studies were not channel (Albesa et al., 2011; Guo et al., 2012). Furthermore, miR-1 designed to address this issue. It is also possible that indirect directly regulates human KCNJ2, a channel that maintains cardiac effects, such as reductions in Nedd4-mediated ubiquitylation of resting potential (Yang et al., 2007). These findings suggest that DEVELOPMENT

872 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Fig. 6. Regulation of Nedd4 by miR-1 is evolutionarily conserved. (A) (Top) Luciferase activity is shown in transfected C2C12 (murine myoblast) cells of a reporter construct containing the 3′UTR of murine Nedd4l (left) or a deletion of the predicted miR-1 binding element (right). *P≤0.05. (Bottom) A schematic showing base pairing of Nedd4l mRNA with miR-1. Blue font indicates the miR-1 seed sequence. Blue box indicates deleted residues that disrupted miR-1 sensitivity, as tested in the above panels. (B) qRT-PCR demonstrating changes in miR-1 expression postnatally. miR-1 levels were normalized to Sno202. **P<0.01; P, postnatal day. (C) (Left) Representative western blot of whole heart-cell lysates from wild-type postnatal day (P) 2 and P21 mice. (Right) Quantification of band intensity for Nedd4L protein normalized to GAPDH. *P<0.05. n, number of hearts. (D) Relative expression levels determined by RNA sequencing of Nedd4l transcripts in wild-type versus miR-1−/− hearts at embryonic stage 18 (E18). *P<0.05. (E) (Left) Western blot of Nedd4L in heart lysates from postnatal day 2 (P2) from wild-type or m-miR-1−/− mice (n=3 per genotype). GAPDH served as a loading control. (Right) Quantification of band intensity for Nedd4L protein normalized to GAPDH. *P<0.05. n, number of hearts. the regulation of murine Nedd4l by miR-1 contributes to some of Images were processed using Adobe Photoshop CS6 and the white set-point the electrophysiological abnormalities seen in miR-1 null mice function was used for background lightening. (Heidersbach et al., 2013; Wei et al., 2014; Zhao et al., 2007). It Drosophila would be interesting to determine whether Nedd4L is dysregulated embryonic imaging Embryos were collected and stained with various antibodies as previously in the heart after an infarction or under ischemic conditions, when described (Han and Olson, 2005). Rabbit anti-Dmef2 was used at 1:1000 miR-1 is upregulated and fatal cardiac dysrhythmias are common. dilution. Cy3, Cy5 or biotin-conjugated secondary antibodies (from Jackson Labs) were used. Adult flies were dissected and fixed for 10 min in 4% MATERIALS AND METHODS paraformaldehyde in phosphate-buffered saline (PBS). Alexa Fluor R555 Fly stocks phalloidin was obtained from Thermo Fisher. Mouse anti-pericardin Drosophila lines were obtained from Bloomington Stock Center (NIH antibody (EC11) was used at 1:500 dilution, followed by Cy3-conjugated P40OD018537). The following fly lines were generously provided by secondary antibodies (Jackson Labs). Confocal imaging was performed with G. Tear (King’s College London, UK): Nedd4T119FS, Nedd4N121FS and a Zeiss ApoTome.2 microscope using a 20× Plan-Apochromat 0.8 N.A. UAS-Nedd4 (Myat et al., 2002). W1118 flies served as wild-type controls. air objective. For quantitative comparisons of intensities, common settings Transgenes were overexpressed with the UAS-GAL4 system (Brand and were chosen to avoid oversaturation. ImageJ Software Version 1.49 was used Perrimon, 1993). The following GAL4 and UAS lines were used: dpp-GAL4 to process images. (Bloomington Stock Center) and UAS-miR-1 (C. Kwon, Johns Hopkins University, Baltimore, MD, USA) (Kwon et al., 2005). The Ellipse allele Drosophila larval and adult heart imaging (ElpB1) was provided by G. Rubin (HHMI Janelia Research Campus, VA, Larvae and adult flies were dissected and fixed for 10 min in 4% USA) (Baker and Rubin, 1989). paraformaldehyde in phosphate-buffered saline (PBS). Alexa Fluor 555 phalloidin was obtained from Thermo Fisher. Mouse anti-pericardin Wing imaging antibody (EC11) was used at 1:500 dilutions, followed by Cy3- Adult Drosophila were anesthetized with CO2, placed in isopropanol for conjugated secondary antibodies (Jackson Labs). Confocal imaging was 1 min and euthanized. Flies were air dried, their wings were removed and performed with a Zeiss ApoTome.2 microscope using a 20× Plan- embedded in Canada Balsam (Sigma), and covered with a glass coverslip. Apochromat 0.8 N.A. air objective. For quantitative comparisons of Images were taken on a LeicaMZ16F microscope at 5× magnification with a intensities, common settings were chosen to avoid oversaturation. A Leica DFC310FX camera and the Leica Application Suite (LAS) program. minimum of 6 larvae or adults per genotype were visualized. ImageJ DEVELOPMENT

873 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Software Version 1.49 was used for image processing. For quantitative Statistics comparisons of cardiac muscle fiber density, cardioblast cell numbers and The rates of genetic enhancement or truncation of long vein 3 (L3) were pericardin deposition we analyzed six control flies and six flies of each analyzed with the chi-square test. Mean, s.d. and s.e.m. were calculated for experimental genotype. Sample size determinations were based upon luciferase assays. qRT-PCR of relative miR-1 levels were performed with extensive previous experience of mutant fly heart morphological analysis. GraphPad Prism (version 6.0) for t-tests. Differences between two means were assessed by two-tailed t-tests, unless otherwise stated. Error bars Fly survival assay represent s.e.m. Null hypotheses were rejected at P<0.05. On day 6 after egg laying, Drosophila larvae were transferred from 25°C to 29°C to enhance the temperature-sensitive UAS-transgene expression. Acknowledgements Adult male flies were subsequently maintained at 29°C in vials containing We thank Gary Howard, Stephen Ordway, Adam Richman, Crystal Herron and 15 or animals. Sixty flies were assayed per genotype. Bethany Taylor for editorial assistance, Karen Carver-Moore and Lei Liu for mouse colony support, and Joshua Urrutia for technical support.

Luciferase assay Competing interests Approximately 200 base pairs surrounding the predicted miR-1-target sites The authors declare no competing or financial interests. in the untranslated regions (UTRs) of Nedd4 or Nedd4l were amplified 1118 directly from cDNA generated from W flies or wild-type mouse hearts, Author contributions respectively, and subcloned with XbaI into the PGL3 (Promega) firefly I.N.K. designed and performed the Drosophila wing assay and conducted western luciferase vector at the 3′ end of the reporter gene. Correct insertion was blotting on wild-type mouse hearts. J.-y.Z. and Z.H. performed the misexpression confirmed by sequencing. analysis, rescue studies of the fly heart and fly mortality studies. A.H., K.N.I. and D.S. For transfection of the C2C12 murine myoblast cell line (ATCC) with generated the miR-1-null mice. A.H. performed the western blot on the miR-1-null luciferase constructs and a Renilla normalization vector, we used heart cell lysates and the luciferase assays. I.S.K. and B.I.G. obtained and Lipofectamine 2000 (Life Technologies) according to the manufacturer’s determined the miR-1 levels from wild-type murine hearts. Z.H. and I.N.K. wrote the manuscript. instructions. Briefly, cells in 12-well plates were transfected at 60% confluency and analyzed 20 h later. Each well received 3 μlof Funding Lipofectamine 2000, 800 ng of PGL3-Target and 200 ng of Renilla This work was supported by a Mentored Research Training Grant from the vector. Experimental wells received 10 pmol of miR-1 mimic (Ambion/ Foundation for Anesthesia Education and Research (I.S.K.), by the National Life Technologies), and control wells received 10 pmol of a non-targeting Institutes of Health (NIH) (R01 HL057181 to D.S., K08 HL079260, R01HL090801 control mimic (Ambion/Life Technologies). and R01DK098410), by the American Heart Association (AHA-0630178N to Z.H.) Firefly and Renilla luciferase activities in lysates were quantified with the and by a Basil O’Connor Starter Scholar Research Award (5-FY10-489) from the Dual Luciferase Reporter Assay kit (Promega) and a Victor 1420 Multilabel March of Dimes Foundation to I.N.K. Deposited in PMC for release after 12 months. Counter (PerkinElmer). Firefly luciferase values were normalized to Renilla to control for transfection efficiency. Supplementary information Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.140368.supplemental RNA sequencing Whole hearts from embryonic day 18 (E18) wild-type and miR-1 double- References knockout mice were isolated and analyzed as described previously Abriel, H., Kamynina, E., Horisberger, J.-D. and Staub, O. (2000). Regulation of (Heidersbach et al., 2013). the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4. FEBS Lett. 466, 377-380. Quantitative PCR Albesa, M., Grilo, L. S., Gavillet, B. and Abriel, H. (2011). Nedd4-2-dependent Postnatal hearts were obtained from C57BL/6J pups after timed matings, ubiquitylation and regulation of the cardiac potassium channel hERG1. J. Mol. Cell. Cardiol. 51, 90-98. with n=3 at each time point. Animals were sacrificed by CO2 asphyxiation Baker, N. E. and Rubin, G. M. (1989). Effect on eye development of dominant followed by decapitation. Hearts were snap frozen in liquid nitrogen and mutations in Drosophila homologue of the EGF receptor. Nature 340, 150-153. subsequently transferred to Trizol (ThermoFisher Scientific) and Brand, A. H. and Perrimon, N. (1993). Targeted as a means of homogenized in a Bullet Blender using 2 mm ZrSiO beads, according altering cell fates and generating dominant phenotypes. Development 118, to the manufacturer’s instructions (Next Advance). RNA was isolated 401-415. using the Direct-zol RNA MiniPrep kit (catalog number R2052) from Bruce, M. C., Kanelis, V., Fouladkou, F., Debonneville, A., Staub, O. and Rotin, D. (2008). Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif Zymo Research. cDNA was prepared using a High Capacity cDNA located within its HECT-domain. Biochem. J. 415, 155-163. Reverse Transcription Kit (catalog number 4368814) and miR-1 TaqMan Casso, D. J., Biehs, B. and Kornberg, T. B. (2011). A novel interaction between probes (catalog number 4427975) from Thermo Fisher. miR-1 levels were hedgehog and Notch promotes proliferation at the anterior-posterior organizer of normalized to SnoRNA-202 (catalog number 427975) according to the Drosophila wing. Genetics 187, 485-499. manufacturer’s recommendations. Mice of both sexes were housed Chen, H., Ross, C. A., Wang, N., Huo, Y., MacKinnon, D. F., Potash, J. B., under the UCSF ‘Assurance of Compliance with PHS Policy on Simpson, S. G., McMahon, F. J., DePaulo, J. R., Jr and McInnis, M. G. (2001). ’ NEDD4L on human chromosome 18q21 has multiple forms of transcripts and is a Humane Care and Use of Laboratory Animals by Awardee Institutions , homologue of the mouse Nedd4-2 gene. Eur. J. Hum. Genet. 9, 922-930. and their use was approved by the UCSF Institutional Animal Care and de Celis, J. F. and Garcıa-Bellido,́ A. (1994). Roles of the Notch gene in Drosophila Use Committee. wing morphogenesis. Mech. Dev. 46, 109-122. Drechsler, M., Schmidt, A. C., Meyer, H. and Paululat, A. (2013). The conserved Western blots ADAMTS-like protein lonely heart mediates matrix formation and cardiac tissue integrity. PLoS Genet. 9, e1003616. Hearts were isolated from mice on postnatal days 2 and 21 (P2, P21) and rinsed Easow, G., Teleman, A. A. and Cohen, S. M. (2007). Isolation of microRNA targets with 1× PBS. The tissue was resuspended in RIPA buffer and disassociated in a by miRNP immunopurification. RNA 13, 1198-1204. Bullet Blender using 2 mm ZrSiO beads according to the manufacturer’s Fu, J., Akhmedov, D. and Berdeaux, R. (2013). The short isoform of the ubiquitin instructions (Next Advance). After clarification and sonication, the lysates were ligase NEDD4L is a CREB target gene in hepatocytes. PLoS ONE 8, e78522. loaded onto a 4-12% SDS-PAGE gel (Biorad), blotted onto polyvinylidene Guo, J., Wang, T., Li, X., Shallow, H., Yang, T., Li, W., Xu, J., Fridman, M. D., fluoride (PDVF) membranes by standard protocols, and probed with primary Yang, X. and Zhang, S. (2012). Cell surface expression of human ether-a-go-go- antibodies against GAPDH (Abcam; 1:5000) and Nedd4L (Bethyl A302- related gene (hERG) channels is regulated by caveolin-3 protein via the ubiquitin ligase Nedd4-2. J. Biol. Chem. 287, 33132-33141. 512A; 1:2000). Blots were visualized and quantified with a Licor Odyssey Han, Z. and Olson, E. N. (2005). Hand is a direct target of Tinman and GATA factors system and fluorescently conjugated secondary antibodies (Licor, Lincoln, during Drosophila cardiogenesis and hematopoiesis. Development 132, NE), according to the manufacturer’s instructions. 3525-3536. DEVELOPMENT

874 RESEARCH ARTICLE Development (2017) 144, 866-875 doi:10.1242/dev.140368

Hasson, P., Egoz, N., Winkler, C., Volohonsky, G., Jia, S., Dinur, T., Volk, T., Persaud, A., Alberts, P., Amsen, E. M., Xiong, X., Wasmuth, J., Saadon, Z., Courey, A. J. and Paroush, Z. (2005). EGFR signaling attenuates Groucho- Fladd, C., Parkinson, J. and Rotin, D. (2009). Comparison of substrate dependent repression to antagonize Notch transcriptional output. Nat. Genet. 37, specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays. 101-105. Mol. Syst. Biol. 5, 333. Heidersbach, A., Saxby, C., Carver-Moore, K., Huang, Y., Ang, Y.-S., de Jong, Rotin, D. and Kumar, S. (2009). Physiological functions of the HECT family of P. J., Ivey, K. N. and Srivastava, D. (2013). microRNA-1 regulates sarcomere ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 10, 398-409. formation and suppresses smooth muscle gene expression in the mammalian Rotin, D., Staub, O. and Haguenauer-Tsapis, R. (2000). Ubiquitination and heart. Elife 2, e01323. endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of Ingham, R. J., Gish, G. and Pawson, T. (2004). The Nedd4 family of E3 ubiquitin ubiquitin-protein ligases. J. Membr. Biol. 176, 1-17. ligases: functional diversity within a common modular architecture. Oncogene 23, Rougier, J.-S., van Bemmelen, M. X., Bruce, M. C., Jespersen, T., Gavillet, B., 1972-1984. Apotheloz, F., Cordonier, S., Staub, O., Rotin, D. and Abriel, H. (2005). Itoh, M., Kim, C.-H., Palardy, G., Oda, T., Jiang, Y.-J., Maust, D., Yeo, S.-Y., Molecular determinants of voltage-gated sodium channel regulation by the Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al. (2003). Mind bomb is a Nedd4/Nedd4-like proteins. Am. J. Physiol. Cell Physiol. 288, C692-C701. ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Sakata, T., Sakaguchi, H., Tsuda, L., Higashitani, A., Aigaki, T., Matsuno, K. and Dev. Cell 4, 67-82. Hayashi, S. (2004). Drosophila Nedd4 regulates endocytosis of notch and Ivey, K. N., Muth, A., Arnold, J., King, F. W., Yeh, R.-F., Fish, J. E., Hsiao, E. C., suppresses its ligand-independent activation. Curr. Biol. 14, 2228-2236. Sokol, N. S. and Ambros, V. (2005). Mesodermally expressed Drosophila Schwartz, R. J., Conklin, B. R., Bernstein, H. S. et al. (2008). MicroRNA microRNA-1 is regulated by Twist and is required in muscles during larval regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem growth. Genes Dev. 19, 2343-2354. Cell 2, 219-229. van Bemmelen, M. X., Rougier, J.-S., Gavillet, B., Apothéloz, F., Daidie, D., Jespersen, T., Membrez, M., Nicolas, C. S., Pitard, B., Staub, O., Olesen, S. P., Tateyama, M., Rivolta, I., Thomas, M. A., Kass, R. S., Staub, O. et al. (2004). Baro, I. and Abriel, H. (2007). The KCNQ1 potassium channel is down-regulated Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated by ubiquitylating of the Nedd4/Nedd4-like family. Cardiovasc. Res. 74, ubiquitination. Circ. Res. 95, 284-291. 64-74. Wei, Y., Peng, S., Wu, M., Sachidanandam, R., Tu, Z., Zhang, S., Falce, C., Sobie, Keating, M. T. and Sanguinetti, M. C. (2001). Molecular and cellular mechanisms E. A., Lebeche, D. and Zhao, Y. (2014). Multifaceted roles of miR-1s in of cardiac arrhythmias. Cell 104, 569-580. repressing the fetal gene program in the heart. Cell Res. 24, 278-292. King, I. N., Qian, L., Liang, J., Huang, Y., Shieh, J. T. C., Kwon, C. and Wilkin, M. B., Carbery, A.-M., Fostier, M., Aslam, H., Mazaleyrat, S. L., Higgs, J., Srivastava, D. (2011). A genome-wide screen reveals a role for microRNA-1 in Myat, A., Evans, D. A. P., Cornell, M. and Baron, M. (2004). Regulation of notch modulating cardiac cell polarity. Dev. Cell 20, 497-510. endosomal sorting and signaling by Drosophila Nedd4 family proteins. Curr. Biol. Krzystanek, K., Rasmussen, H. B., Grunnet, M., Staub, O., Olesen, S.-P., Abriel, 14, 2237-2244. H. and Jespersen, T. (2012). Deubiquitylating USP2 counteracts Yang, B. and Kumar, S. (2010). Nedd4 and Nedd4-2: closely related ubiquitin- Nedd4-2-mediated downregulation of KCNQ1 potassium channels. Heart protein ligases with distinct physiological functions. Cell Death Differ. 17, 68-77. Rhythm 9, 440-448. Yang, B., Lin, H., Xiao, J., Lu, Y., Luo, X., Li, B., Zhang, Y., Xu, C., Bai, Y., Wang, Kwon, C., Han, Z., Olson, E. N. and Srivastava, D. (2005). MicroRNA1 influences H. et al. (2007). The muscle-specific microRNA miR-1 regulates cardiac cardiac differentiation in Drosophila and regulates Notch signaling. Proc. Natl. arrhythmogenic potential by targeting GJA1 and KCNJ2. Nat. Med. 13, 486-491. Acad. Sci. USA 102, 18986-18991. Zhao, Y., Ransom, J. F., Li, A., Vedantham, V., von Drehle, M., Muth, A. N., Myat, A., Henry, P., McCabe, V., Flintoft, L., Rotin, D. and Tear, G. (2002). Tsuchihashi, T., McManus, M. T., Schwartz, R. J. and Srivastava, D. (2007). Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking cell surface levels of the roundabout receptor. Neuron 35, 447-459. miRNA-1-2. Cell 129, 303-317. DEVELOPMENT

875